Ben Bradley

Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not

Summary. In postnatal life, mesenchymal stem cells (MSC) self‐replicate, proliferate and differentiate into mesenchymal tissues, including bone, fat, tendon, muscle and bone marrow (BM) stroma. Possible clinical applications for MSC in stem cell transplantation have been proposed. We have evaluated the frequency, phenotype and differentiation potential of MSC in adult BM, cord blood (CB) and peripheral blood stem cell collections (PBSC). During culture, BM MSC proliferated to confluence in 10–14 d, maintaining a stable non‐haemopoietic phenotype, HLA class‐1+, CD29+, CD44+, CD90+, CD45–, CD34– and CD14 through subsequent passages. Using the colony forming unit fibroblasts assay, the estimated frequency of MSC in the BM nucleated cell population was 1 in 3·4 × 104 cells. Both adipogenic and osteogenic differentiation of BM MSC was demonstrated. In contrast, CB and PBSC mononuclear cells cultured in MSC conditions for two passages produced a population of adherent, non‐confluent fibroblast‐like cells with a haemopoietic phenotype, CD45+, CD14+, CD34–, CD44–, CD90– and CD29–. In paired experiments, cultured BM MSC and mature BM stroma were seeded with CB cells enriched for CD34+. Similar numbers of colony‐forming units of granulocytes–macrophages were produced by MSC‐based and standard stroma cultures over 10 weeks. We conclude that adult BM is a reliable source of functional cultured MSC, but CB and PBSC are not.

Endothelial cell precursors are normal components of human umbilical cord blood

Endothelial cells are part of the normal bone marrow stroma. We have previously shown human umbilical cord blood (UCB) does not produce stroma in standard long‐term cultures. Highly enriched (93–98%) UCB CD34+ cells were cultured for 6 weeks with interleukin‐2 and conditioned medium from the 5637 carcinoma cell line (n = 4). The resulting ‘fibroblast like’ cells were shown to be endothelial by expression of von Willebrand factor (VWF), ICAM‐1 (CD54), E‐selectin (CD62E) and PECAM (CD31). Endothelial monolayers seeded with CD34+ UCB cells supported expansion of colony forming cells and CD34+ cells. We conclude that endothelial cell precursors circulate in UCB, and may be derived from the CD34+ cell fraction.

Impact of obstetric factors on cord blood donation for transplantation

Recent reports have shown that low nucleated cell dose significantly decreases survival after cord blood transplantation. Prior to starting clinical cord blood banking we investigated the impact of obstetric factors on cell dose and volume of cord blood donations. Cord blood was obtained from 114 normal full‐term deliveries. Mean volume collected was 93.5 ml, mean total nucleated cell count (TNC) was 13.1 × 108. Statistical analysis was by backwards stepwise regression. Significant factors affecting nucleated cell yield were volume of blood collected (P < 0.001), length of gestation (P < 0.0001), time from delivery of the infant to cord clamping (P = 0.018) and total length of labour (P = 0.002). In clinical cord blood banking we have successfully used these findings for pre‐collection assessment of placentae. Out of 476 cord blood donations subsequently collected for banking, only 29 (6.1%) have been discarded due to low volume. The mean TNC of the 409 banked units following volume reduction was 10.1 × 108. Despite careful optimization of collection, processing and storage techniques, cell dose still limits cord blood transplantation to smaller recipients.

Cytokine Expansion Culture of Cord Blood CD34+ Cells Induces Marked and Sustained Changes in Adhesion Receptor and CXCR4 Expressions

Recent studies have demonstrated defective bone marrow homing of hematopoietic stem cells after cytokine expansion culture. Adhesion receptors (ARs) are essential to the homing process, and it is possible that cytokine culture modulates AR expression. We studied changes in expression of very late antigen‐4 (VLA‐4), VLA‐5, L‐selectin, leukocyte function‐associated antigen‐1 (LFA‐1), CD44, and the stromal cell‐derived factor‐1 (SDF‐1) receptor, CXCR4, during cytokine culture of cord blood (CB) CD34+ cells.

Is in vitro expansion of human cord blood cells clinically relevant

Peripheral blood recovery after cord blood (CB) transplantation is delayed compared with marrow. Expansion of CB haemopoietic cells has been investigated with the aim of reducing cytopenia following transplantation. Mature cells, post-progenitors, progenitors and long-term culture-initiating cells (LTC-IC) from expansion products may contribute to peripheral blood recovery. We investigated the increase in total nucleated cells, colony-forming cells (CFC), CD34+ cells and LTC-IC by limiting dilution after a 14 day culture of CB CD34+ cells (5 × 103/ml) with SCF, IL-3, IL-6, GM-CSF and G-CSF all at 10 ng/ml. On average, nucleated cells increased 2500-fold, CD34+ cells 39-fold and CFU-GM 49-fold with maintenance of BFU-E. The more primitive LTC-IC expanded on average 2.5-fold but effects on long-term marrow-repopulating cells (LTRC) during culture are unknown. A practical application of in vitro expansion of CB might be to expand a 20% aliquot of a CB donation and infuse the remainder unmanipulated. This could provide a 5- to 7-fold increase in progenitor cells, an estimated 1570-fold increase in post-progenitor cells and maintenance of LTC-IC compared to an untreated donation. Combined with in vivo post-transplant growth factor therapy this could prompt early peripheral blood recovery after CB transplantation, without significant loss of LTC-IC or donor lymphocytes.

The CD34+CD38neg population is significantly increased in haemopoietic cell expansion cultures in serum-free compared to serum-replete conditions: Dissociation of phenotype and function

Expansion of haemopoietic stem cells is proposed to combat graft failure in adult recipients following cord blood (CB) transplantation. Cultures are traditionally performed in medium containing FCS, but to transfer expansion to the clinic, ‘good manufacturing practice’ (GMP) standards are required. This study evaluated expansion cultures in culture bags and serum-free (SF) conditions, to comply with GMP, by analysing sub-populations of CD34+ cells, colony-forming cells (CFC) and long-term culture initiating cells (LTC-IC). CD34+cell analysis has previously been used to measure clonogenic capacity and the CD34+CD38neg surface phenotype to measure primitive cell numbers. In this study, comparison of expansion in serum-replete medium with that in SF conditions demonstrated a lack of expression of CD38 on CD34+ cells in the absence of serum. These findings must be considered in clinical studies using in vitro expansion in SF conditions, and the CD34+CD38neg phenotype should not be used to confirm maintenance, or expansion, of primitive progenitor cells. Bone Marrow Transplantation (2001) 27, 365–371.

Longevity in vitro of human CD4+ T helper cell clones derived from young donors and elderly donors, or from progenitor cells: Age-associated differences in cell surface molecule expression and cytokine secretion

The effectiveness of the adaptive immune system reliesupon extensive proliferation of an initially smallnumber of antigen-specific T cells. At the end of asuccessful response, the majority die by apoptosis anda small minority joins the memory cell pool. Uponre-challenge with antigen, these memory cells mustagain undergo clonal expansion in order to mediate aneffective response. Thus, T cells are subjected tomarked proliferative stress which may result in clonalexhaustion due to replicative senescence. In othersystems made up of rapidly proliferating cells (e.g. inthe gut) individual clones are identical and arereplaced at the end of their lifespan bydifferentiation from a stem cell reservoir. However,because of the unique clonal distribution of antigenreceptors on T cells, mere replacement with other Tcells is not sufficient to maintain the integrity ofthe system. Moreover, the very source of new T cellsdecreases with age (due to thymic involution).Therefore, the adaptive immune system may be uniquelysusceptible to the deleterious effects of replicativesenescence. Particularly in humans, in vivo studies ofthe behaviour of individual T-cell clones in the bodyis difficult. However, T-cell longevity, measured asproliferative capacity in terms of populationdoublings, can be usefully modelled at the clonallevel in vitro. This paper discusses the surprisinglylittle that is known about the average longevity,variation between clones, and the maximal longevity ofhuman T cells under clonal culture conditions invitro. From our own studies, we show that averagelifespan of human T cells is as little as 17 PD;however, established clones reach 35 PD on average,with maximum longevity generally in the region of 60–80 PD, regardless of the source of the cloned cells.Expression of surface molecules in general did notdiffer strikingly between young and old donors, butthe frequency of clones secreting IL-10, and theamount secreted per clone was higher in the elderlythan in the young. Conversely, the frequency of clonessecreting IL-6 and the amount secreted per clone washigher in the young.

Diagnosis of sickle-cell disease with a universal heteroduplex generator

A new diagnostic technique, universal heteroduplex generator screening, has been developed to detect homozygosity and heterozygosity for sickle-cell disease. Since it is a polymerase chain reaction-based technique, it may be used to detect haemoglobin S and haemoglobin C genotypes in adults, neonates, or from coelocentesis during the first 10 weeks of pregnancy. beta-globin gene nucleotide sequences are amplified by the polymerase chain reaction, and are heteroduplexed with a beta-globulin universal heteroduplex generator. Haemoglobins S and C mutations are identified by characteristic polyacrylamide minigel banding patterns. The technique is simple and rapid to do, even by nonspecialist laboratories, and is applicable to large-scale screening for haemoglobin S and C mutations as well as prenatal diagnosis of sickle cell disease.

Cryopreserved Human Bone Marrow Stroma is Fully Functional In vitro

Human marrow long‐term culture (LTC) enables maintenance of both stromal and haemopoietic elements of normal bone marrow (NBM) in vitro for 4–6 months. Stroma‐based cultures are critical for quantitation of long‐term culture initiating cells (LTC‐IC), the most primitive human haemopoietic cells measurable in vitro. Supply of NBM can be sporadic, and up to 3–4 weeks in culture is required for stromal maturity. Stroma availability for experimental purposes can therefore be limited. Efforts to produce transformed human and transfected murine stromal cell lines comparable to NBM stroma have had some success. As an alternative, we investigated cryopreserved NBM and cryopreserved preformed stroma. Function of cryopreserved and control fresh NBM stroma was similar when evaluated for up to 12 weeks in LTC. We have also demonstrated that stroma derived from cryopreserved NBM or preformed cryopreserved NBM stroma can sustain third‐party haemopoiesis as efficiently as fresh NBM stroma in LTC. Batched cryopreserved stroma is a convenient, rapidly available, source of functional stroma which avoids the logistic difficulties and lack of standardization associated with stroma from fresh NBM. This important advance will enhance the use of stroma‐based LTC in studies of human haemopoiesis.

In vitro expansion of cord blood does not prevent engraftment of severe combined immunodeficient repopulating cells.

Summary. This study aimed to assess the potential of human cord blood (CB) cells to engraft in the xenogenic non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model after in vitro expansion culture. We also studied the quality of human haemopoiesis arising from the transplantation of fresh or expanded cells in this model. Cord blood CD34+ cells were cultured for 3, 7 or 10 d with stem cell factor, Flt3, thrombopoietin, interleukin 3 (IL‐3), IL‐6 and granulocyte colony‐stimulating factor, all at 10 ng/ml in serum‐replete conditions. Transplantation of mice with fresh CB containing 3 × 104 CD34+ cells and 1–2 SCID repopulating cells (SRC) resulted in a median of 7·4% (0·4%‐76·8%) human engraftment. When mice received the expanded product of 1–2 SRC, the ability to repopulate NOD/SCID mice was maintained even after 10 d of in vitro culture. Serial dilution of the expanded cells suggested that in vitro expansion had increased SRC numbers two‐ to fourfold. Expanded SRC produced long‐term culture‐initiating cells, clonogenic cells and CD34+ cells in the same proportions as fresh cells after successful engraftment. Therefore, expanded SRC were able to differentiate in the same way as fresh SRC. There was a trend towards lower levels of engraftment when d 7 cultured cells were transplanted (median engraftment 0·8%, range 0·0–24·0%) compared with 1–2 fresh SRC. Our data suggest that this is owing to reduced proliferation of cultured cells in vivo. By utilizing limiting numbers of CB SRC, we confirmed that the engraftment potential of SRC in the NOD/SCID model was preserved after in vitro expansion. Furthermore, dilution experiments strongly suggest two‐ to fourfold expansion of SRC in vitro. These studies are relevant for developing clinical stem cell expansion strategies.